Project description:Understanding the molecular basis of how the tick adapts to feed on different animal hosts is central to understanding tick and tick-borne disease (TBD) epidemiology. Tick adaptation to feed on vertebrate hosts is regulated by tick secretion of multiple tick saliva proteins (TSPs) and other molecules that regulate tick feeding. This study was initiated to determine if ticks such as Ixodes scapularis and Amblyomma americanum that are adapted to feed on multiple hosts utilized the same sets of proteins to accomplish feeding on all hosts. Our data suggest that ticks of the same species differentially express proteins when feeding on diffent hosts. SDS-PAGE and silver staining analysis revealed unique protein eletrophoretic profile in saliva of Ixodes scapularis and Amblyomma americanum that were stimulated to start feeding on different hosts: rabbits, humans, and dogs. LC-MS/MS sequencing and pairwise analysis of proteins in saliva of I. scapularis and A. americanum ticks that were non-stimulated and those that were stimulated to feed on rabbits, dogs, or humans identified TSPs that were unique to each treatment and those that were common. Overal, we identified a total of 276 and 340 non-redundant I. scapularis and A. americanum TSPs, which we have classified into 28 functional classes that include secreted conserved proteins (unknown functions), proteinase inhibitors, lipocalins, extracellular matrix/cell adhesion, heme/iron metabolism, signal transduction and immunity-related proteins being the most predominant in saliva of unfed ticks. With exception of Rhipicephalus microplus, anti-tick vaccine research relies on feeding lab animals. Data here suggest that lab animal data could result in prioritizing irrelevant targets as some tick genes are unique to ticks fed on lab animals. This study provides the platform that could be utilized to identify relevant target anti-tick vaccine antigens, and will facilitate early stage tick feeding research.
Project description:Background Human monocytotropic ehrlichiosis is an emerging life-threatening zoonosis caused by obligately intracellular bacterium, Ehrlichia chaffeensis. E. chaffeensis is transmitted by the lone star tick, Amblyomma americanum, and replicates in mononuclear phagocytes in mammalian hosts. Differences in the E. chaffeensis transcriptome in mammalian and arthropod hosts are unknown. Thus, we determined host-specific E. chaffeensis gene expression in human monocyte (THP-1) and in Amblyomma and Ixodes tick cell lines (AAE2 and ISE6) using a whole genome microarray. Methodology/Principal Findings The majority (~80%) of E. chaffeensis genes were expressed during infection in human and tick cells. There were few differences observed in E. chaffeensis gene expression between the vector Amblyomma and non-vector Ixodes tick cells, but extensive host-specific and differential gene expression profiles were detected between human and tick cells, including higher transcriptional activity in tick cells and identification of gene subsets that were differentially expressed in the two hosts. Differentially and host-specifically expressed ehrlichial genes encoded major immunoreactive tandem repeat proteins (TRP), the outer membrane protein (OMP-1) family, and hypothetical proteins that were 30–80 amino acids in length. Consistent with previous observations, high expression of p28 and OMP-1B genes was detected in human and tick cells, respectively. Notably, E. chaffeensis genes encoding TRP32 and TRP47 were highly upregulated in the human monocytes and expressed as proteins; however, although TRP transcripts were expressed in tick cells, the proteins were not detected in whole cell lysates demonstrating that TRP expression was post transcriptionally regulated. Conclusions/Significance Ehrlichia gene expression is highly active in tick cells, and differential gene expression among a wide variety of host-pathogen associated genes occurs. Furthermore, we demonstrate that genes associated with host-pathogen interactions are differentially expressed and regulated by post transcriptional mechanisms.
Project description:Background Human monocytotropic ehrlichiosis is an emerging life-threatening zoonosis caused by obligately intracellular bacterium, Ehrlichia chaffeensis. E. chaffeensis is transmitted by the lone star tick, Amblyomma americanum, and replicates in mononuclear phagocytes in mammalian hosts. Differences in the E. chaffeensis transcriptome in mammalian and arthropod hosts are unknown. Thus, we determined host-specific E. chaffeensis gene expression in human monocyte (THP-1) and in Amblyomma and Ixodes tick cell lines (AAE2 and ISE6) using a whole genome microarray. Methodology/Principal Findings The majority (~80%) of E. chaffeensis genes were expressed during infection in human and tick cells. There were few differences observed in E. chaffeensis gene expression between the vector Amblyomma and non-vector Ixodes tick cells, but extensive host-specific and differential gene expression profiles were detected between human and tick cells, including higher transcriptional activity in tick cells and identification of gene subsets that were differentially expressed in the two hosts. Differentially and host-specifically expressed ehrlichial genes encoded major immunoreactive tandem repeat proteins (TRP), the outer membrane protein (OMP-1) family, and hypothetical proteins that were 30–80 amino acids in length. Consistent with previous observations, high expression of p28 and OMP-1B genes was detected in human and tick cells, respectively. Notably, E. chaffeensis genes encoding TRP32 and TRP47 were highly upregulated in the human monocytes and expressed as proteins; however, although TRP transcripts were expressed in tick cells, the proteins were not detected in whole cell lysates demonstrating that TRP expression was post transcriptionally regulated. Conclusions/Significance Ehrlichia gene expression is highly active in tick cells, and differential gene expression among a wide variety of host-pathogen associated genes occurs. Furthermore, we demonstrate that genes associated with host-pathogen interactions are differentially expressed and regulated by post transcriptional mechanisms. A microarray (4-plex) study using E. chaffeensis cultivated in each cell line (THP-1, AAE2 and ISE6), three biological replicates/cell line. For each cell line, RNA was also extracted from uninfected cells (negative controls) and was processed similar to the infected cells; these samples were used for background subtraction during data analysis.
Project description:One of the most important vectors of the Brazilian Spotted Fever, the tick Amblyomma aureolatum in Brazil was used in this study. We laboratorial controlled the infection of adult females of A. aureolatum with the virulent brazilian strain Taiacu of Rickettsia rickettsii. The group of ticks was divided into 2 testing groups, group 1 (G1) composed of adult females incubated at 25°C for 3 days and group 2 (G2) composed of adult females incubated at 35°C for 3 days. Right after incubation of both groups, ticks were individually dissected and all internal organs were transferred to RNAlater® Solution (Life Technologies) until gDNA and total RNA simultaneously isolation. A total of 14 ticks of each group were analyzed in two biological replicates (7 ticks each). Dye-swap was also applied to construct the technical replicate of each biological sample total RNA from both experimental samples (G1 and G2) was used for hybridization to dual channel arrays. Two biological replicates were used for each experimental group.
Project description:One of the most important vectors of the Brazilian Spotted Fever, the tick Amblyomma aureolatum in Brazil was used in this study. We laboratorial controlled the infection of adult females of A. aureolatum with the virulent brazilian strain Taiacu of Rickettsia rickettsii. The group of ticks was divided into 2 testing groups, group 1 (G1) composed of adult females incubated at 25°C for 3 days and group 2 (G2) composed of adult females incubated at 35°C for 3 days. Right after incubation of both groups, ticks were individually dissected and all internal organs were transferred to RNAlater® Solution (Life Technologies) until gDNA and total RNA simultaneously isolation. A total of 14 ticks of each group were analyzed in two biological replicates (7 ticks each). Dye-swap was also applied to construct the technical replicate of each biological sample